Human umbilical vein endothelial cells (HEC) in culture synthesize and release a plasminogen activator inhibitor which is found in the culture medium in two forms, an active form which binds to and inactives tissue plasminogen activator and urokinase and a latent form which lacks anti-activator activity in its native state but which becomes active upon treatment with SDS. Studies from this laboratory suggest that the inhibitor is synthesized and released from HEC in its active form and converts to the latent form in the extracellular environment. Using the human inhibitor from cultured HEC, I propose to investigate the mechanism by which the transition from active to latent inhibitor occurs and to determine whether the rate of this conversion can be regulated. The physiochemical properties of the two forms will be studied and compared employing isoelectric focusing, sucrose and cesium chloride centrifugation and molecular exclusion chromatography. A correlation between the active to latent inhibitor transition and changes in molecular properties will be sought. Studies will be performed with purified active inhibitor to determine whether this transition is catalyzed by a second component in the culture medium or if it is an intrinsic characteristic of the molecule occurring independently of its environment. The observed association between the inhibitor and the endothelial cell matrix will be further investigated by immunofluorescence and binding studies. These studies will further define a potentially important mechanism by which plasminogen activator inhibitor activity is controlled in vivo.